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Star Republic: Guide for Biologists

RNA purification --- acid phenol:chloroform extraction

1. To RNA sample add equal volume of acid phenol:chloroform (5:1, pH 4.7). Vortex.

Phenol must be water-saturated first. Check pH. If pH is too high, lower with acetic acid. Alternatively, lower the sample pH by adding 1/10 volume of 2 M sodium acetate (pH 4).

2. Centrifuge for 10 minutes, and remove the aqueous phase to a new tube. The aqueous phase contains RNA.

3. Add 1/10 volume of 5 M ammonium acetate and equal volume of isoproponal.

4. Mix well. Precipitate for at least 15 minutes at -20 C.

5. Pellet RNA by centrifugation at maximum speed for at least 15 minutes at 4 C in a microcentrifuge.

6. Carefully remove supernatant. And wash with 70% ethanol.

9. Air dry and resuspend in a suitable buffer. Store at -20 C or -80 C.